For both protein chemistry (Nilsson et al. (1992) Curr. Opin. Struct. Biol. 2: 569-575; LaVallie and McCoy (1995) Curr. Opin. Biotechnol. 6: 501-506; Uhlen and Moks (1990) Methods Enzymol. 185: 129-143) and organic synthesis applications (Sears and Wong (1996) Biotechnol. Prog. 12: 423-433; Faber (1997) Biotransformations in Organic Synthesis: 3rd ed. Springer-Verlag: Heidelberg; Roberts (1993) Preparative Biotransformations; Wiley: New York: 1993) it is desirable to have available a diverse toolbox of inexpensive proteases with high selectivity and diverse substrate preferences. To date the most extensively exploited class of enzymes in organic synthesis applications have been the hydrolases. Among these, the serine proteases have received considerable attention due, in part, to their often exquisite stereo-, region, and chemo-selectivities (Sears and Wong (1996) Biotechnol. Prog. 12: 423-433; Faber (1997) Biotransformations in Organic Synthesis: 3rd ed. Springer-Verlag: Heidelberg; Roberts (1993) Preparative Biotransformations; Wiley: New York: 1993; Moree et al. (1997) J. Am. Chem. Soc. 119: 3942-3947).
While over 3000 enzymes have now been reported, of which many are proteases, significantly fewer of the latter are available inexpensively from commercial sources (Faber (1997) Biotransformations in Organic Synthesis: 3rd ed. Springer-Verlag: Heidelberg; Roberts (1993) Preparative Biotransformations; Wiley: New York: 1993; Moree et al. (1997) J. Am. Chem. Soc. 119: 3942-3947; Jones (1986) Tetrahedron 42: 3351-3403). Furthermore, since wild type enzymes do not accept all substrate structures of synthetic interest, it is attractive to contemplate the tailoring of a readily available protease in order to expand their substrate specificities in a controlled manner with the ultimate goal of creating any desired specificity at will.
In this regard, the goal of specificity alteration of enzymes has already been targeted by several different approaches. For example, site-directed mutagenesis (Perona and Craik (1995) Protein Sci. 4: 337-360) and random mutagenesis (Arnold (1998) Acc. Chem. Res. 31(3): 125-131) have been employed to tailor enzyme specificity and have permitted some insights into the electrostatic (Wells et al. (1987) Proc. Natl. Acad. Sci. USA, 84: 5167-5171; Wells et al. (1987) Proc. Nat. Acad. Sci. USA, 84: 1219-1223; Wells and Estell (1988) TIBS 13: 291-297; Bott et al. (1987) Pages 139-147 In: Biotech. Agric. Chem.; Lebanon, Mumma, Honeycutt, Duesing, eds.; Vol. ACS Symp. Ser. 334; Russell et al. (1987) J. Mol. Biol. 193: 803-813; Ballinger et al. (196) Biochemistry 33: 13579-13585), steric (Rheinnecker et al. (1994) Biochemistry 33: 221-225; Rheinnecker et al. (1993) Biochemistry 32(5): 1199-1203; Sørensen et al. (1993) Biochemistry 32: 8994-8999; Estell et al. (1986) Science 233: 659-663; Takagi et al. (1996) FEBS Lett. 395: 127-132; Takagi et al. (1997) Protein Eng. 10(9): 985-989), and hydrophobic (Estell et al. (1986) Science 233: 659-663; Wangikar et al. (1995) Biochemistry 34(38): 12302-12310; Bech et al. (1993) Biochemistry 32: 2845-2852) factors which govern enzyme-substrate interactions. However, the structural variations within these approaches are limited to the 20-natural amino acids. Consequently, biosynthetic methods have recently been developed to introduce unnatural amino acids into proteins (25. Cornish et al. (1995) Angew. Chem. Int. Ed. Eng. 34: 621-633; Parsons et al. (1998) Biochemistry 37: 6286-6294; Hohsaka et al. (1996) J. Am. Chem. Soc. 118(40): 9778-9779). Unnatural functionalities have also been incorporated by chemical modification techniques (Kuang et al. (1996) J. Am. Chem. Soc. 118: 10702-10706; Ory et al. (1998) Protein. Eng. 11(4): 253-261; Peterson: E. B.; Hilvert: D. Biochemistry 34: 6616-6620; Suckling: C. J.; Zhu: L.-M. Bioorg. Med. Chem. Lett. 3: 531-534; Rokita and Kaiser (1986) J. Am. Chem. Soc. 108: 4984-4987; Kokubo et al. (1987) J. Am. Chem. Soc. 109: 606-607; Radziejewski et al. (1985) J. Am. Chem. Soc. 107: 3352-3354). Generally, however, unnatural amino acid mutagenesis approach is not yet amenable to large scale preparations, and chemical modification alone is insufficiently specific.